Anticoagulant Rodenticide Toxicity to Non-Target Wildlife Under Controlled Exposure Conditions Barnett A
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University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln USDA National Wildlife Research Center - Staff U.S. Department of Agriculture: Animal and Plant Publications Health Inspection Service 2018 Anticoagulant Rodenticide Toxicity to Non-target Wildlife Under Controlled Exposure Conditions Barnett A. Rattner U.S. Geological Survey, [email protected] F. Nicholas Mastrota USEPA Follow this and additional works at: https://digitalcommons.unl.edu/icwdm_usdanwrc Part of the Life Sciences Commons Rattner, Barnett A. and Mastrota, F. Nicholas, "Anticoagulant Rodenticide Toxicity to Non-target Wildlife Under Controlled Exposure Conditions" (2018). USDA National Wildlife Research Center - Staff Publications. 2103. https://digitalcommons.unl.edu/icwdm_usdanwrc/2103 This Article is brought to you for free and open access by the U.S. Department of Agriculture: Animal and Plant Health Inspection Service at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in USDA National Wildlife Research Center - Staff ubP lications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Chapter 3 Anticoagulant Rodenticide Toxicity to Non- target Wildlife Under Controlled Exposure Conditions Barnett A. Rattner and F. Nicholas Mastrota 1 Introduction Our knowledge of the toxicity of anticoagulant rodenticides (ARs) can be traced to investigations of Karl Paul Link and colleagues on “bleeding disease” in cattle, the eventual isolation of dicoumarol from moldy sweet clover, synthesis of this causative agent, and its application as a therapeutic anticoagulant in clinical medicine in 1941 (Link 1959). The notion of a coumarin-based rodenticide as a better “mouse-trap” occurred to Link in 1945 while reviewing laboratory chemical and bioassay data. By 1948, the highly potent compound number 42, warfarin, was promoted as a rodenti- cide (Link 1959; Last 2002). Through laboratory studies and clinical use of warfarin (Coumadin), a detailed understanding of the mechanism of action and toxicity of warfarin and related ARs (Fig. 3.1) unfolded in the decades that followed. Our understanding of AR toxicity has been principally derived from an array of biochemical through whole animal studies. Structure-activity relationship models indicate that AR potency (i.e., toxicity in rodents) is related to the length and hydro- phobicity of the side chain in the vicinity of carbon 13 (Fig. 3.2), with the most active compounds having greater volume and bulky lipophilic groups in this activity domain (Thijssen 1995; Domella et al. 1999). At the molecular level, both coumadin- and indandione-based ARs inactivate vitamin K epoxide reductase (VKOR), a membrane protein present in the endoplasmic reticulum of liver and other tissues. Catalytic activity of VKOR is required for the reduction of vitamin K epoxide and vitamin K to form vitamin K hydroquinone (Fig. 3.3). This biologically-active B.A. Rattner (*) U.S. Geological Survey, Patuxent Wildlife Research Center, BARC East-Building 308, 10300 Baltimore Avenue, Beltsville, MD 20705, USA e-mail: [email protected] F.N. Mastrota U.S. Environmental Protection Agency, Office of Chemical Safety and Pollution Prevention, Washington, DC 20460, USA © Springer International Publishing AG 2018 45 N.W. van den Brink et al. (eds.), Anticoagulant Rodenticides and Wildlife, Emerging Topics in Ecotoxicology 5, DOI 10.1007/978-3-319-64377-9_3 46 B.A. Rattner and F.N. Mastrota First-generation hydroxycoumarins Warfarin Coumachlor 81-81-2 81-82-3 Coumafuryl Coumatetralyl 117-52-2 5836-29-3 Intermediate-generation indandiones Chlorophacinone Diphacinone Pindone 3691-35-8 82-66-6 83-26-1 Fig. 3.1 Class, compound, Chemical Abstracts Service Number and structure of 12 anticoagulant rodenticides (From: https://www.ncbi.nlm.nih.gov/pccompound) hydroquinone is required for γ-glutamyl carboxylation of clotting factors. Inhibition of VKOR by ARs limits the formation of vitamin K hydroquinone resulting in under-carboxylation of clotting factors II, VII, IX and X (Furie et al. 1999) that do not assemble on cell surfaces to form a clot. It is believed that ARs bind tightly to the proposed warfarin-binding site of VKOR at tyrosine residue 135 in close proximity to the active site (cysteines 132 and 135) of this 163 amino acid enzyme (Tie and Stafford 2008). Notably, some point mutations can impede AR binding and thus confer resistance in target pest species (Boyle 1960; Pelz et al. 2005). Once the fully-functional clotting factors are cleared from the blood, the des-γ carboxyl dysfunctional clotting factors no longer support hemostasis. Hemorrhage may ensue spontaneously or can be triggered by traumatic events. Coagulopathy may be accompanied by anemia, hypovolemic shock, altered tissue perfusion, organ 3 Anticoagulant Rodenticide Toxicity to Non-target Wildlife Under Controlled… 47 Second-generation hydroxycoumarins Brodifacoum Bromadiolone Difenacoum 56073-10-0 28772-56-7 56073-07-5 Difethialone Flocoumafen 104653-34-1 90035-08-8 Fig. 3.1 (continued) dysfunction, and necrosis. Overt signs of intoxication include bruising, bleeding, blood in droppings and urine, pallor, and other signs not specific to coagulopathy (e.g., asthenia, ataraxia, labored breathing, immobility). The proximate cause of death may seemingly be unrelated to AR poisoning, but in fact ultimately triggered by AR-residues and coagulopathy. In addition to impaired blood clotting, some ARs have been shown to increase membrane permeability, affect other vitamin K-dependent proteins, growth factors, and signal transduction (reviewed in Rattner et al. 2014a). Notably, large doses of indandiones can cause toxicity and result in death independent of coagulopathy (Kabat et al. 1944), probably by impairing cellular energy generation through the uncoupling of oxidative phosphorylation Fig. 3.2 Structure of the first-generation anticoagulant rodenticides warfarin and diphacinone, and the second- generation anticoagulant rodenticide brodifacoum, illustrating side chains (red) of the activity domain (*) in vicinity of carbon 13 (Modified with permission from Rattner et al. 2014a, Copyright 2015 American Chemical Society) (Color figure online) Fig. 3.3 Vitamin K cycle illustrating anticoagulant rodenticide (AR) sensitive vitamin K epoxide reductase (VKOR) reactions and a warfarin-insensitive VKOR that reduces vitamin K to the biologically- active vitamin K hydroquinone. Without adequate vitamin K hydroquinone, γ-glutamyl carboxylase (critical reaction circled in green) lacks substrate to adequately carboxyl- ate clotting factors II, VII, IX and X (Reprinted with permission from Rattner et al. 2014a, Copyright 2015 American Chemical Society) 3 Anticoagulant Rodenticide Toxicity to Non-target Wildlife Under Controlled… 49 (van den Berg and Nauta 1975). Numerous controlled exposure studies have docu- mented in vitro biochemical effects, and in vivo physiological, pharmacological and whole organism responses in domesticated species and to a lesser degree captive wildlife (reviewed in IPCS 1995; Joermann 1998; Rattner et al. 2014a), and much is known from clinical use and accidental poisoning incidents in humans (Watt et al. 2005). Recently, a proposed adverse outcome pathway, identifying the molecular initiating/anchoring event, and established and plausible linkages associated with toxicity through individual and even population levels, has been developed for non- target predatory birds and mammals (Fig. 3.4) (Rattner et al. 2014a) The use of vertebrate pesticides, and specifically ARs, requires detailed toxico- logical knowledge and regulatory evaluation to ensure a compound does not pose an unacceptable risk to non-target biota and the environment (Eason et al. 2010). The review and approval process takes into account economic, social and environmental costs and benefits (Eason et al. 2010). An integral component of this process is the generation of toxicity data for non-target wildlife. These data are used to examine the potential hazard and risk associated with direct bait ingestion and consumption of AR-exposed prey by non-target species. For purposes of AR registration, much of these data are generated using standardized toxicity testing methods. However, additional research on AR absorption, distribution, metabolism, pharmacokinetics and underlying mechanism of action is often undertaken to more fully evaluate and explain interspecific differences in toxicity. The generation of these data usually entails in vivo testing in species maintained in captivity using various exposure scenarios. This chapter will principally focus on data generated from such studies in terrestrial wildlife (mammals, birds and reptiles) or domesticated surrogate species used to predict effects in non-target wildlife. 1.1 Standardized Tests, Their Limitations and Implications As terrestrial wildlife may be exposed by direct consumption of AR-containing bait and/or by predation or scavenging on exposed or poisoned rodents, standard- ized tests have focused on the dietary route of exposure. Notably, exposure path- ways have yet to be clearly elucidated for aquatic species. Standardized testing protocols allow regulators to compare the toxicities of various chemicals to ter- restrial wildlife and examine the range of species sensitivities to a particular chemical. In the wildlife- pesticide regulatory arena, the most commonly used endpoint for toxicity is mortality because of its definitive nature. The two most commonly conducted standardized